Protein-protein interaction is one of the mechanisms of signal transduction processes. One such process involves non-receptor type protein-tyrosine kinases (PTKs) of the Src family, which signal in normal and transformed cells. In recent years, much of the attention has been concentrated on certain specific regions of PTKs, in particular three structural domains, termed SH2 (SH stands for Src homology), SH3, and PH (pleckstrin homology). In addition to the Src family of proteins, these domains are present in a wide variety of proteins implicated in signal transduction processes.
Src Homology 2 and 3 (SH2 and SH3) domains are globular protein modules present in a large variety of functionally distinct proteins (Koch et al., 1991, Science 252: 668; Musacchio et al., 1992, FEBS Lett. 307: 55; Mayer et al., 1993, Trends Cell Biol. 3: 8). They mediate binding events that control the activity and localization of many proteins involved in the transmission of signals from the cell surface to the nucleus. SH2 domains bind to phosphorylated tyrosine residues (Matsuda et al., 1991, Mol. Cell. Biol. 11: 1607; Waksman et al., 1992, Nature 358: 646; Cantley et al., 1991, Cell 64: 281). The specificity of these phosphorylation-dependent interactions is determined by sequences adjacent to the phosphorylated tyrosines and has been extensively analyzed (Songyang et al., 1993, Cell 72: 767; Songyang et al., 1994, Mol. Cell. Biol. 14: 2777; Lee et al., 1994, Structure 2: 423; Birge et al., 1993, Science 262: 1522 (and references therein). In contrast to the abundant data on SH2 domain specificity, the binding specificity of SH3 domains is less well understood.
The presence of SH3 domains in proteins of lower eucaryotes such as S. cervisiae is indicative of the functional importance of SH3 domains throughout evolution. Despite significant sequence diversity, structural comparisons of many SH3 domains show similar folds (Musacchio et al., 1992, Nature 359: 851). The first SH3 binding protein was isolated through its ability to bind to the AblSH3 domain and the sequence responsible for this interaction was localized to a ten amino acid proline-rich fragment (Cicchetti et al., 1992, Science 257: 803; Ren et al., 1993, Science 259: 1157). It has been suggested that proline-rich SH3-binding motifs have a structure similar to that of a polyproline II helix (Lim et al., 1994, Structural Biol. 1: 221). In such a helix each turn consists of three amino acids. Amino acids that are three positions apart are therefore oriented coplanar in space. Ultrastructural analysis of the interaction of a high affinity, proline-rich binding peptide with the PI3Kp85-a-SH3 domain demonstrated that two coplanar proline residues form contacts with two grooves on the surface of the SH3 domain (Yu et al., 1994, Cell 76: 933). These grooves contain highly conserved aromatic amino acids and are spaced approximately at a distance of one turn of a polyproline II helix.
The Crk proteins belong to a family of proteins that consist almost entirely of SH2 and SH3 domains, with little intervening sequence. This family presently includes v-Crk (Mayer et al., 1988, Nature 332: 272), two forms of c-Crk proteins, c-Crk-I and c-Crk-II (Reichman et al., 1992, Cell Growth Diff. 3: 451; Matsuda et al., 1992, Mol. Cell Biol. 12: 3482), CRKL (ten Hoeve et al., 1993, Oncogene 8: 2469), Grb2/ASH (Matuoka et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9015; Lowenstein et al., 1992, Cell 70: 431; Suen et al.,1993 Mol. Cell. Biol. 13: 5500) and its homologs Sem5 (Clark et al., 1992, Nature 356: 340) and Drk (Olivier et al., 1993, Cell 73: 179; Simon et al., 1993, Cell 73: 169), Grb3-3 (Fath et al., 1994, Science 264: 971), and Nck (Lehmann et al., 1990 Nucl. Acids Res. 18: 1048). Expression of v-Crk or elevated expression of c-Crk-I leads to cell transformation and increased cellular phosphotyrosine levels (Mats, 16, 18, 28, 29), but the biological role of c-Crk proteins is currently unknown. Since these proteins lack apparent catalytic domains, their function probably lies in their ability to bind specific proteins via their SH2 and SH3 domains. Proteins that interact with the CrkSH2 domain via phosphorylated tyrosine residues have been first identified in cells transformed by v-Crk or v-Src (4, 29, 30). The CrkSH2 domain was shown to preferentially bind phosphotyrosyl-peptides that contain a pTyr-X-X-Pro motif (7 ,30). Such a high affinity binding motif is generated upon phosphorylation of c-Crk-II by c-Abl in the spacer region between the two CrkSH3 domains. Binding of the CrkSH2 to this phosphotyrosine residue may regulate c-Crk functions (31).
Src homology 3 (SH3) domains are present on many functionally diverse proteins that are involved in the response of cells to external stimuli (Musacchio et al., 1994, Mol. Cell Biol. 14:5495; Kuriyan and Cowburn, 1993, Curr. Op. Struct. Biol. 3:828-37). The ability of the SH3 domains to bind to short, specific, linear, proline-rich sequences (Cicchetti et al., 1992, Science 257:803) within their binding partners has important functional consequences. SH3 domains may control the cellular localization of their binding partners (Bar-Sagi et al., 1993, Cell 74:83-91); Rotin et al., 1994, EMBO J. 13:4440), determine the substrate specificity of enzymes (Feller et al., 1994, EMBO J. 13:2341-2351) or modulate the catalytic activity of SH3-containing (Liu et al., 1993, Mol. Cell Biol. 13:5225; Mayer and Baltimore, 1994, Mol. Cell Biol. 14:2883), as well as SH3-binding proteins (Gout et al., 1993, Cell 75:25; Pleiman et al., 1994, Science 263:1609). Despite the abundance of SH3 domains within signalling proteins and their potential biological importance, only a few systems are known where SH3 domains play a direct role in physiological or pathological processes. These include genetic systems that demonstrated the crucial role of complex formation between the adaptor protein, Grb2 and the Guanine-nucleotide exchange factor, Son of Sevenless (SOS) (Olivier et al., 1993, Cell 73:179), and two human diseases that involve loss of function mutations of SH3 domains Rawlings et al., 1993, Science 261:358) or their binding sequences (Cheng et al., 1994, Proc. Natl. Acad. Sci. USA 91:8152).
Identification of two proline-rich motifs in the SH3 binding proteins, 3BP1 and 3BP2 was first achieved by expression library screening with the AblSH3 domain (Cicchetti et al., supra). Mutagenesis of the 3BP-1 sequence pointed to the proline residues at positions 2, 5 and 10 that are crucial in the binding to the AblSH3 domain (Ren et al., 1993, Science 259:1157). Proline-rich motifs were subsequently identified in known SH3 targets (Seedorf et al., 1994, J. Biol. Chem. 269:16009; Ren et al., 1994, Genes Dev. 8:783; Tanaka et al., 1994, Proc. Natl. Acad. Sci. USA 91:3443), and found to bind to several SH3 domains with variable affinities (Liu et al., 1993, supra; Gout et al., 1993, supra; Chardin et al., 1993, Science 260:1338). Only one highly selective interaction, the binding of a proline-rich sequence in p47.sup.phox to the C-terminal p.sub.67.sup.l phox SH3 domain has been reported (Finan et al., 1994, J. Biol. Chem. 269:13752). More recently, new SH3 binding sequences that are not necessarily found in known proteins have been delineated by screening of combinatorial peptide (Yu et al., 1994, Cell 76:933) and phage display libraries (Cheadle et al., 1994, J. Biol. Chem. 269:24034; Sparks et al., 1994, J. Biol. Chem. 269:23853) and provide valuable information about affinity and specificity requirements for SH3 domains.
Structural and mutational analyses of SH3 domain-peptide complexes from Sem-5, p85.alpha., Abl and Fyn (Lim and Richards, 1994, Nature Structural Biology 1:221; Booker et al., 1993, Cell 73:813; Musacchio et al., 1994, Nature Structural Biology 1:546; Yu et al., 1994, supra) revealed molecular details of interactions between SH3 domains and proline-rich sequences. The SH3 domain core is composed of two perpendicular, antiparallel, 3-stranded .beta.-pleated sheets. The hydrophobic surface contains shallow grooves formed by highly conserved aromatic amino acids which contact the proline-rich peptides. This area interacts with two coplanar proline residues from the SH3 binding motif, which forms a polyproline type II helix (Williamson, 1994, Biochem. J. 297:245-260). A second set of coplanar amino acids in positions next to the two proline residues contacts the hydrophobic surface as well as the highly variable RT and n-Src loops that emerge from the core. Despite the contacts of these four amino acids, the affinities of SH3 domains to their ligands are in the micromolar range (Yu et al., 1994, supra).
The general features of SH3 structure and the interaction with proline-rich peptides are now understood (Kudyan & Cowburn, 1993, Curr. Op. Struct. Biol., 3:828-837, Saraste & Musacchio, 1994, Nature Struct. Biol., 1:835-837). The structures of a number of uncomplexed SH3 domains have been determined and have established a highly conserved fold consisting of two anti-parallel .beta. sheets packed against each other (reviewed in (Kudyan & Cowburn, 1993, Curr. Op. Struct. Biol., 3:828-837)). The discovery that SH3 domains bind to sequences that contain multiple prolines with characteristic PXXP motifs (Cicchetti et al., 1992, Science, 257, 803-806, Ren et al., 1993, Science, 259:1157-1161) was followed by the proposal that the SH3 surface could recognize a polyproline type II helix (Lim & Richards, 1994, Nature Struct. Biol. 1:221-225) and by the determination of the three-dimensional structures of peptide complexes of the p85 SH3 domain by NMR (Yu et al., 1994, Cell, 76:933-945) and the Abl and Fyn SH3 domains by X-ray crystallography, Musacchio et al., 1994, Nature Struct. Biol., 1:546-551). These first structures of SH3 complexes revealed that a conserved set of residues on the SH3 surface provide hydrophobic interactions with sidechains presented by the polyproline type II helix.
In each of these structures, the peptide bound in the same orientation ("plus" orientation). Four papers have recently appeared describing the interaction of proline-rich peptides with the Src SH3 domain (Feng et al., 1994, Science, 266:1241-1247) and with SH3 domains of mammalian Grb2 (Goudreau et al., 1994, Nature Struct. Biol., 1:898-907, Terasawa et al., 1994, Nature Struct. Biol, 1:891-897) and its C. elegans homolog Sem-5 (Lim et al., 1994, Nature, 372:375-379). The structures of Src SH3 domain bound to two peptides obtained by selection from a combinatorial library revealed that while one peptide bound in the "plus" orientation, the other bound in the opposite direction ("minus" orientation). The three structures of Grb2/Sem-5 SH3 domains show that the Sos peptides bind in the "minus" orientation. The ability of different prolin-rich peptides to bind in opposite orientations to the same binding sites on the SH3 surface can be understood in terms of the symmetry of the polyproline helix and the specific electrostatic and hydrophobic packing interactions observed in these complexes, and analyses of these structures have led to the enunciation of general rules for prolin-rich peptides binding to SH3 domains (Feng et al., 1994, Science, 266:1241-1247, Lim et al., 1994, Nature, 372:375-379).
In these recent structures of the Src (Feng et al., 1994, Science, 266:1241-1247, Lim et al.) and Grb2/Sem5 (Goudreau et al., 1994, Nature Struct. Biol., 1:898-907, Terasawa et al., 1994, Nature Struct. Biol, 1:891-897, Lim et al., 1994, Nature, 372:375-379) peptide complexes, the orientation of the peptide is determined primarily by electrostatic interactions between arginine residues In the peptide and acidic residues that are localized at one end of the peptide binding surface of the SH3 domains. Many SH3 domains have acidic residues in this region. For example, most SH3 domains have a glutamate or aspartate residue corresponding to Glu 172 in Sew-5 that is observed to hydrogen bond to the arginine residue in the Sos peptide (Lim et al., 1994, Nature, 372:375-379). Likewise, the central interactions via PXXP motifs involve highly conserved hydrophobic residues in the SH3 domains. Consequently, the basis for sequence discrimination between prolin-rich peptides and particular SH3 domains requires further elaboration.
The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention.